JPS6318960B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION It has become possible to produce proteins by recombinant DNA methods, in particular the production of insulin A chain and insulin B chain by this type of method [Goeddel et al, Proc.
Nat'l.Acad.Sci.USA, 76 , 106-110 (1979)], A
There is a great need for efficient methods for producing insulin by combining insulin and B chains. A typical prior art method for producing insulin by combining A and B chains uses stable S-
The A and B chains are used in the sulfonate form. In general, the S-sulfonates of the A and B chains, separately or together, are combined with the corresponding -SH
The compound is reduced, usually using a large excess of thiol reducing agent. The products are isolated from the reducing reaction medium and, when reduced separately, are brought together and contacted with an oxidizing medium, such as air, to combine the A and B chains and form insulin. Examples of this method include Du et al, Scientia Sinica,
10, 84-104 (1961); Wilson et al, Biochim.
Biophys. Acta, 62 , 483-489 (1962); Du et al.
Scientia Sinica, 14 , 229-236 (1965); Kung
et al, Scientia Sinica, 15 , 544-561 (1966);
Kexue Tongbao (Republic of China), 17 , 241
~277 (1966); and Markussen, J. Acta
Paediatrica Scandinavica, Suppl., 270 , 121−
126 (1977). A modification of this process is to reduce the A chain S-sulfonate and then react the reduced A chain with the B chain S-sulfonate in an oxidizing atmosphere. For example, Katsoyannis et al, Proc. Nat.
Acad.Sci. (USA), 55 , 1554-1561 (1966),
Katsoyannis, Science, 154 , 1509−1514
(1966); Katsoyannis et al, Biochemistry,
6, 2642-2655 (1967); US Patent No. 3420810
and Jentsch, Journal of Chromatography,
See 76, 167–174 (1973). Another improvement is to partially oxidize the -SH compound of the A chain to form a disulfide between the cysteine residues of A-6 and A-11, and then add this compound to the -SH compound of the B chain or the -SH compound of the B chain. There is a method of oxidation with S-sulfonate. for example,
Belgian Patent No.676069 and Zahn et al.
See Liebigs Ann.Chem., 691 , 225-231 (1966). Each of the above prior art methods has one thing in common. It consists of two independent, sequential steps: S-sulfonate -
Insulin is produced by the ring element to SH and subsequent oxidation to -S-S-. Dixon et al, Nature, 188 , 721–724 (1960)
suggested the use of a thiol reducing agent and air oxidation to convert A and B chain S-sulfonates to insulin in a single solution. The description is very incomplete and the yield is based only on the activity of the recovered product and is 1-2%.
However, Dixon said that Proc.Intern.Congr.
Endecrinol.2nd London1964, 1207−1215 (1965)
I added some explanations, especially in the table on page 1211,
The reactions reported in earlier publications reveal that ring element and oxidation were carried out in separate steps. Unlike the prior art methods described above, both reduction and oxidation reactions are performed in a single step and in a single solution to produce insulin or insulin analogs from S-sulfonated A and B chains in attractive yields. It has been found that it is possible under certain reaction conditions to obtain The present invention relates to such a method. That is, the present invention relates to a method for producing insulin or an insulin analog by combining the A chain of insulin or an insulin analog with the B chain of insulin or an insulin analog, and the gist thereof is as follows:
A chain S-sulfonate, a B chain S-sulfonate, and a thiol reducing agent in an aqueous medium, (1)
The pH is about 8 to about 12, and (2) the total protein concentration is about
0.1 to about 50 mg/ml and (3) all the - present in the S-sulfonate forms of A and B chains.
SSO ^- about 0.4 to about 2.5 - for each of the _three
Insulin or insulin analogs are prepared by mixing in the presence of an amount of thiol reducing agent capable of providing SH groups and maintaining the mixture at a temperature of about 0°C to about 25°C in an atmosphere provided with an oxygen source. The point is that it generates. The present invention relates to an effective one-step, single-solution process for producing insulin or insulin analogs from the corresponding S-sulfonated A and B chains. The term "insulin" as used herein, of course, refers to natural insulin from humans, cows, pigs, sheep, fish, birds, etc., as well as hybrid insulins in which the A chain of some species and the B chain of other species are combined. The term "insulin analog" refers to a wide variety of proteins consisting of A and B basic chains with the same sequence as natural insulin and a half-cystine residue.
These analogs differ from native insulin in that one or more amino acid residues are substituted, added, omitted, or modified, but they retain at least some of their disulfide bond alignment and insulin-like activity. It is something that is kept. Examples of such "insulin analogs" include [N-formyl-
Gly ¹ -A] insulin, desamino-A ¹ -insulin, [sarcosine ¹ -A] insulin, [L-
Alanine ¹ -A] insulin, [D-alanine ¹ -
A] insulin, [isoasparagine ²¹ -A] insulin, [D-asparagine ²¹ -A] insulin, [arginine ²¹ -A] insulin, [asparaginamide ²¹ -A] insulin, [sarcosine ¹ -
A-Asparagine ²¹ -A] insulin, [norleucine ² -A] insulin, [threonine ⁵ -A]
Insulin, [leucine ⁵ -A] insulin, [phenylalanine ¹⁹ -A] insulin, [D-tyrosine ¹⁹ -A] insulin, [tyrosine ¹⁸ -A, asparagine ¹⁹ -A, arginine ²¹ -A] insulin, des [ B ^28-30 -tripeptide] insulin,
Des[B ^27-30 -tetrapeptide] insulin, des[B ^26-30 -pentapeptide] insulin, des[B ^27-30 -tetrapeptide, tyrosinamide ²⁶ -
B] insulin, des[B ^26-30 -pentapeptide, phenylalaninamide ²⁵ -B] insulin, des[B ^1-4 -tetrapeptide] insulin,
Examples include des[B ^1-5 pentapeptide] insulin, [lysine ²² -B] insulin, [leucine ⁹ -B] insulin, [leucine ¹⁰ -B] insulin, and des [phenylalanine ¹ -B] insulin.
These and other insulin analogs have been described in the literature. For example, Blundell, T., et al.
Advances in Protein Chemistry, 26 , 330−
362, Academic Press, NY, NY (1972);
Katsoyannis, PG, Treatment of Early
Diabetes, 319-327, Plenum Publishing Corp.
(1979); Geiger, R., Chemiker Zeitung
Reprint 100, 111-129, Dr. Hu¨thig,
Publisher, Heidelberg, W.Germany (1976);
Brandenburg, D. et al., Biochem.J., 125 , 51
-52 (1971). Although the method of the invention is broadly applicable to the production of insulin and insulin analogues, it is more preferably used for the production of natural insulin, even more preferably for the production of human, bovine or porcine insulin, and most preferably for the production of human insulin. It will be done. In carrying out the method of the invention, the production of insulin or insulin analogues by combining the A and B chains can be carried out within a very wide range with respect to the relative proportions of one chain to the other. Of course, this binding reaction is necessarily constrained by the quantitatively less abundant chain of both AB chains. In any case, the weight ratio of A chain to B chain is not essential, but is usually about 0.1:1 to about
The ratio is 10:1. In carrying out the method of the invention, it is preferred that the weight ratio of A and B chains be from about 1:1 to about 3:1. Furthermore, within this range, certain ranges have been found to be particularly advantageous for producing certain insulins. That is, when A chain and B chain are combined to produce bovine insulin, the ratio of A chain:B chain is about 1.4:1.0 to about
Preferably, the ratio is 1.8:1.0. The preferred range for porcine insulin is about 1.0:1.0 to about 1.4:1.0
It is. For the production of human insulin, the preferred range is about 1.8:1.0 to about 2.2:1.0. Another parameter of importance for optimal performance of the method of the invention is the protein concentration in the reaction medium. The method can be successfully performed over a wide range of protein concentrations. however,
The protein concentration in the reaction medium is generally approximately 0.1
The concentration ranges from 50 mg/ml to 50 mg/ml. Preferably, the protein concentration ranges from about 2 to 20 mg/ml. Within this latter range, the optimal protein concentration will vary depending on the type of insulin being produced. Thus, for porcine insulin, a protein concentration of about 8 to about 16 mg/ml is preferred, while for the production of human or bovine insulin, the preferred range is about 3 to about 8 mg/ml. The method of the invention is carried out in an aqueous medium. The PH of the medium, measured at room temperature, generally ranges from about 8 to about 12. Preferably it is about 9.5 to about 11.0;
Most preferably maintained between about 10.4 and about 10.6. The PH of the medium is maintained in the desired range by adding appropriate buffers. Typical buffers of this type include, for example, glycine, glycylglycine, carbonates, tris(hydroxymethyl)aminomethane, N,N-bis(2-hydroxyethyl)
There are glycine, pyrophosphate, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid and similar substances, which have the effect of controlling the PH within said range. A common and preferred buffering agent is glycine. Buffer concentrations generally range from about 0.001M to about 2M. Preferred concentrations are from about 0.01M to about 1M;
More preferably, it is about 0.01M to about 0.1M. The A and B chains are mixed in a suitable aqueous medium in the presence of a thiol reducing agent. "Thiol reducing agent" means having at least one -SH group, A and B
It is a compound that has the ability to reduce the S-sulfonate group of the chain. Although anything having such properties can be used, more preferred thiol reducing agents are those that cyclize to highly stable compounds in their oxidized form. The thiol reducing agent is A
and in an amount that provides about 0.4 to about 2.5 -SH groups for each -SSO ^- ₃ group present on the B chain, and more preferably about 0.9 to about 1.3 -SH groups for each -SSO ^- ₃ group. Make it exist. Examples of typical thiol reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol, methyl thioglycols, 3-mercapto-1,2-propanediol, mercaptoacetic acid, and 3-mercaptoethanol. These include mercaptopropionic acid, 2-amino-3-mercaptopropionic acid (cysteine), thioglycolic acid and other similar thiol compounds. Preferred thiol reducing agents are dithiothreitol and dithioerythritol, most preferred is dithiothreitol. One of the prerequisites of the process of the invention is that it must be carried out in an atmosphere provided with an oxygen source. This condition is satisfied simply by leaving the reaction mixture open to air. Direct contact methods, such as introducing air or oxygen into the reaction solution, may also be used, but this is not essential. Thus, in general, the process of the present invention is carried out by mixing the A chain S-sulfonate, the B chain S-sulfonate, and the thiol reducing agent at the desired concentration in an aqueous medium having a pH of about 8 to about 12. . The mixture is in open contact with the air and generally for at least about 30 minutes while the chain bonds are fully formed.
Stir gently for a minute. While doing this stirring,
The mixture is generally maintained at a temperature of about 0°C to about 25°C. Preferably, however, the mixture is cooled slowly to maintain it at the lower end of the above range, generally from about 0°C to about 10°C. Once the reaction time has elapsed, the insulin or insulin analog is isolated using any of a variety of methods known in the insulin art.
The most widely used method for purifying insulin is chromatography. These methods are
It can also be easily applied to the recovery of insulin from the method of the present invention. This includes gel filtration and ion exchange chromatography. Furthermore, the purity and activity of the product can be determined by known methods,
For example, polyacrylamide gel electrophoresis, amino acid analysis, insulin radioreceptor assay,
Insulin radioimmunoassay, high performance liquid chromatography (HPLC), ultraviolet spectrum,
It can be assayed by dialysis, rabbit blood glycose assay, etc. Insulin produced by the method of the present invention also includes a hybrid consisting of one type of insulin A chain and another type of insulin B chain. The purpose of the method of the present invention is to appropriately combine the S-sulfonates of the A and B chains, and the structure of each of these chains is the same as that of the A or B chain of insulin or an insulin analog. Not important to the invention method. Although insulin analogues and hybrid insulins (consisting of an A chain of one species and a B chain of another species) can also be produced by the method of the present invention, it goes without saying that insulin having the same structure as natural insulin can be used in combination with such insulins. Preferably, it is produced using an A chain S-sulfonate and a B chain S-sulfonate having the same amino acid sequence. More preferably,
The method of the invention produces porcine, bovine or human insulin, most preferably human insulin. The A and B chains of insulin or insulin analogs are produced by recombinant DNA methods, as described above. It is also produced from natural insulin and by classical peptide synthesis methods including solution or solid phase methods. The A and B chains are stably stored as S-sulfonates. The S-sulfonate feedstock is processed by oxidative sulfitolysis, i.e. A
and by treating the B chain with sodium sulfite in the presence of a mild oxidizing agent, such as sodium tetrathionate. Examples are given below to illustrate the method of the invention. These examples are presented for illustrative purposes only and are not intended to limit the scope of the invention. Example 1 360 mg of porcine A chain S-sulfonate was dissolved in 36 ml of 0.1M glycine buffer (PH10.5), and the pH of the mixture was adjusted to 10.5 with 5N NaOH. 300mg of porcine B chain S-sulfonate in 0.1M glycine buffer (PH10.5)
123.4 mg of dithiothreitol (DTT) was dissolved in 30 ml and the pH of the mixture was adjusted to 10.5 with 5NNaOH.
Dissolve in 4 ml of 0.1M glycine buffer (PH10.5),
The pH of the mixture was adjusted to 10.5 using 0.2 ml of 5NNaOH. A and B chain solutions were kept at room temperature (~25°C).
Then mix the DTT solution in a 100ml vial
After adding 1.91 ml (-SH:-SSO ^- ₃ ratio, 1.04),
The solution was gently stirred with a magnetic stirrer in an open beaker for 20 hours at 4-8°C. Analysis by high performance liquid chromatography (HPLC) revealed that 193.8 mg of insulin was produced, accounting for 29% of the total protein. Adjust 40 ml of this final solution to pH 3.15 with acetic acid, perform gel filtration using an equilibrated Cephadex G-50 (Superfine) column (5 x 200 cm), and 4-8°C using 1M acetic acid. It was eluted with Insulin peak (elution volume, approximately 2450-2700ml)
were pooled and lyophilized to obtain 95 mg of insulin (25% of total protein). This porcine insulin was analyzed by polyacrylamide gel electrophoresis, amino acid analysis,
It was found to be highly pure by insulin radioreceptor assay, HPLC, and rabbit blood glucose lowering test. Example 2 Using 0.01M glycine buffer (PH10.5), solutions of bovine A chain S-sulfonate and bovine B chain S-sulfonate at a concentration of 5 mg/ml were prepared, and each
The pH was adjusted to 10.5 with 5NNaOH. Dissolve 61.7mg of DTT in 4.0ml of 0.1M glycine buffer (PH10.5),
The pH was adjusted to 10.5 with 0.15 ml of 5NNaOH. B chain solution
0.5ml of A chain solution 0.8ml and DTT solution 0.035ml
ml was added at room temperature (~25 °C) (-SH:-SSO ^- ₃
ratio, 0.91). Pour this solution into a 3 ml open vial.
Stirred at 4-8°C for 20 hours. HPLC of this mixture
Analysis showed a yield of 1.96 mg (30% of total protein) of bovine insulin. Example 3 Using 0.1M glycine buffer (PH10.5),
Solutions of porcine A chain S-sulfonate and porcine B chain S-sulfonate at a concentration of mg/ml were prepared, and each solution was adjusted to pH 10.5 with 5N NaOH. 61.7 mg of DTT was dissolved in 2.0 ml of glass distilled water. Add 0.6ml of A chain solution to 0.5ml of B chain solution and
29.25μ of DTT solution was dissolved at room temperature (~25°C) (-SH:-SSO ^- ₃ ratio, 1.00). The mixture was stirred in a 3 ml open vial at 4-8°C for 20 hours. HPLC analysis of this mixture showed a yield of porcine insulin of 3.81
mg (35% of total protein). Example 4 Solutions of human insulin B chain S-sulfonate (derived from pancreas), human insulin A chain S-sulfonate (derived from pancreas and Escherichia coli), and porcine insulin A chain S-sulfonate (derived from pancreas) were
5mg/using 0.1M glycine buffer (PH10.5)
The concentration was adjusted to ml. Each solution was adjusted to pH 10.5 using 5NNaOH. Dissolve 61.7mg of DTT in 4.0ml of 0.1M glycine buffer (PH10.5) and 0.16ml of 5NNaOH.
The pH was adjusted to 10.5. A chain S-sulfonate solution
Add 0.5 ml of B chain S-sulfonate solution to 1.0 ml and
0.05ml of DTT solution was added at room temperature (-SH:-
SSO ^- ₃ ratio, 1.09). All solutions were stored in a cold room (4-8
℃) in an open vial for 20-22 hours. Next, each solution was analyzed by HPLC using pancreatic-derived human insulin as a standard substance for yield calculation. The results are shown in the table below. [Table] Example 5 Human insulin A chain S-sulfonate and human insulin B chain S-sulfonate were dissolved in 0.1 M glycine buffer (PH 10.5) to a concentration of 5 mg/ml, and each was adjusted to PH 10.5 with 5 N NaOH. did.
DTT61.7mg in 0.1M glycine buffer (PH10.5) 4.0
ml, and 0.16 ml of 5NNaOH was added to adjust the pH to 10.5. 1.0 ml of A chain solution was added to 0.5 ml of B chain solution at room temperature, and then 50μ of DTT solution was added (-SH:
-SSO ^- ₃ ratio, 1.09). Pour this solution into an open vial at 4
Stirring at −8° C. for 22 hours followed by HPLC analysis showed a yield of human insulin of 2.58 mg (34% of total protein). Example 6 328 mg of human insulin A chain S-sulfonate
Dissolve in 65.6ml of 0.1M glycine buffer (PH10.5),
The pH was adjusted to 10.5 with 5NNaOH7.5μ. Dissolve 164mg of human insulin B chain S-sulfonate in 32.8ml of 0.1M glycine buffer (PH10.5),
The pH was adjusted to 10.5 with 5NNaOH15μ. DTT61.7mg
Dissolve in 4.0ml of 0.1M glycine buffer (PH10.5),
The pH was adjusted to 10.5 with 5NNaOH160μ. A chain solution and B chain solution at room temperature (~25℃)
Mix DTT solution in a 150ml glass beaker
3.28 ml was added (-SH:-SSO ^- ₃ ratio, 1.09). The open (uncovered) beaker was placed in an ice water bath in a cold room and stirred vigorously for 30 minutes. Store this solution in a cold room (4-
The mixture was further stirred at 8°C for 24 hours. At this point
HPLC analysis showed a yield of 148 mg of human insulin (30% of total protein). Add 25ml of glacial acetic acid to 100ml of this solution, final pH3.15
And so. The entire amount was subjected to gel filtration at 4-8°C using a 5 x 200 cm column of Sephadex G-50 (Superfine) equilibrated with 1M acetic acid and eluted with 1M acetic acid. All of the eluted proteins were lyophilized. The insulin peak (elution volume 2465-2781 ml) was 125 mg, 29.4% of the protein recovered. Part of the above insulin peak (95.5 mg),
0.01M Tris−0.001MEDTA−7.5M Urea−
0.03M sodium chloride buffer (pH at 4°C)
8.5) Dissolved in approximately 9 ml. This mixture was equilibrated with DEAE (diethylaminoethyl) in the same buffer.
Chromatographed on a cellulose ion exchange column 2.5 x 90 cm. Protein elution was carried out using sodium chloride 0.03M (1) and sodium chloride 0.09M (1) at 4-8°C.
) (both in the same buffer) in a concentration gradient using
Then sodium chloride 1M (1) (in the same buffer)
It was eluted with Sephadex G-25 for each peak
After desalting with a (coarse) column and 2% acetic acid, it was freeze-dried. Insulin peak (elution volume 878−1008
ml) was 55.73 mg, which was 84% of the recovered protein. Sample 11.90 mg of insulin (DEAE) peak and add 0.1N hydrochloric acid in a glass centrifuge tube.
Dissolve in 240μ and immediately 0.04% zinc chloride - 0.05M
Zinc insulin crystals were prepared by adding 2.16 ml of sodium citrate-15% acetone solution. Crystallization was carried out at room temperature (~25°C) for 72 hours, the supernatant was removed, and the crystals were washed twice with cold water (PH6.1). For washing, centrifugation was performed at 3°C and 2000 rpm. this crystal
It was dissolved in 0.01N hydrochloric acid and analyzed. The resulting human insulin product was analyzed by polyacrylamide gel electrophoresis (single band), amino acid analysis,
It was determined to be sufficiently pure by insulin radioreceptor assay, insulin radioim assay, HPLC, dansilation and UV spectroscopy. In an assay using rabbits (144 rabbits) according to the US Pharmacopoeia, the titer was 26.3±1.8 units/mg (anhydrous). Example 7 Human (E. coli) [N-formyl-Gly ¹ ] A chain S
- Sulfonate and human (pancreatic) B chain S-sulfonate solution in 0.1M glycine buffer (PH
10.5) to a concentration of 5 mg/ml, and each solution was adjusted to pH 10.5 with 5N NaOH. On the other hand,
DTT61.7mg in 0.1M glycine buffer (PH10.5) 4.0
ml and adjusted to pH 10.5 with 0.16 ml of 5NNaOH. Add 1.0 ml of [N-formyl-Gly ¹ ] A-chain S-sulfonate solution to 0.5 ml of B-chain S-sulfonate solution.
and 0.05 ml of DTT solution were added at room temperature (25°C) (−
SH:−SSO ⁻ ₃ ratio, 1.10). This solution was placed in a 3 ml open vial and stirred in a cold room (4-8°C) for 23 hours. HPLC analysis showed [N-formyl-Gly ¹
-A] Human insulin yield 1.46 mg (of total protein
19.5%). Acidified to PH3.15 using glacial acetic acid and a portion of this solution was gel filtered. For gel filtration, a 1.5 x 90 cm column was filled with Sephadex G-50 (Superfine), equilibrated with 1M acetic acid, and heated at 4 to 8°C.
This was done by elution with [N-formyl-Gly ¹ -A] Human insulin peak (elution volume
87-95 ml) were pooled and a portion was lyophilized. This protein was shown to be sufficiently pure by HPLC and amino acid analysis. The biological activity of [N-formyl-Gly ¹ -A] human insulin evaluated by radioreceptor assay was
It was 17% compared to the standard human insulin. Example 8 Solutions of porcine A chain S-sulfonate and human (E. coli) B chain S-sulfonate were adjusted to a concentration of 10 mg/ml using 0.1 M glycine buffer (PH 10.5). Make an A-B preparation solution by using 2 ml of A chain solution for 1 ml of B chain solution, and add 5NNaOH.
Adjusted to pH 10.5. Dissolve 121.2mg of cysteine in 3.0ml of 0.1M glycine buffer (PH10.5),
The pH was adjusted to 10.5 with 0.35 ml of 5NNaOH. 52 µ of cysteine solution was added to 1.4 ml of the A-B adjustment solution at room temperature (-SH:-SSO ^- ₃ ratio, 0.95). This solution was placed in a 3 ml open vial and stirred for 20 hours at 4-8°C. HPLC analysis showed a human insulin yield of 3.25 mg.
(23.2% of total protein).

Claims (1)

[Scope of Claims] 1. In producing insulin or an insulin analog by combining the A chain of insulin or an insulin analog with the B chain of insulin or an insulin analog, an S-sulfonate of the A chain, a B
The S-sulfonate of the chain and the thiol reducing agent are added in an aqueous medium to provide (a) a pH of 8 to 12, (b) a total protein concentration of 0.1 to 50 mg/ml, and (c) a S-sulfonate of the A and B chains. -0.4 to 2.5 -SH for each of the -SSO ^- ₃ groups present in the -sulfonate
the mixture in the presence of an amount of thiol reducing agent capable of providing the thiol groups and the mixture is maintained at a temperature between 0° C. and 25° C. in an atmosphere provided with an oxygen source to produce insulin or insulin analogs. A manufacturing method characterized by: 2. The method according to claim 1, wherein the A chain S-sulfonate and the B chain S-sulfonate each have the same amino acid sequence as natural insulin. 3. The method according to claim 2, wherein the weight ratio of A chain S-sulfonate to B chain S-sulfonate is 0.1:1 to 10:1. 4. The method according to claim 3, wherein the weight ratio of A chain S-sulfonate to B chain S-sulfonate is 1:1 to 3:1. 5. The method according to claims 1 to 4, wherein the protein concentration is 2 to 20 mg/ml. 6. The method according to claims 1 to 5, wherein the reaction mixture has a pH of 9.5 to 11.0. 7. The method according to claim 6, wherein the reaction mixture has a pH of 10.4 to 10.6. 8. Claim 1, wherein the thiol reducing agent is present in an amount sufficient to provide 0.9 to 1.3 -SH groups for each of all -SSO ^- ₃ groups present in the S-sulfonate forms of A and B chains. 7. The method described in 7. 9. The method according to claim 8, wherein the thiol reducing agent is dithiothreitol or dithioerythritol. 10. The method according to claims 1 to 4, wherein the reaction mixture is maintained at a temperature of 0°C to 10°C. 11 Claims 1 to 10 wherein the insulin produced is bovine, porcine or human insulin
Method described. 12 The insulin to be produced is bovine insulin, and the weight ratio of A chain S-sulfonate to B chain S-sulfonate is 1.4:1.0 to 1.8:1.0,
The method according to claim 11, wherein the protein concentration is 3 to 8 mg/ml. 13 The insulin to be produced is porcine insulin, and the weight ratio of A chain S-sulfonate to B chain S-sulfonate is 1.0:1.0 to 1.4:1.0,
The method according to claim 11, wherein the protein concentration is 8 to 16 mg/ml. 14 The produced insulin is human insulin, and the weight ratio of A chain S-sulfonate to B chain S-sulfonate is 1.8:1.0 to 2.2:1.0,
The method according to claim 11, wherein the protein concentration is 3 to 8 mg/ml.